BACKGROUND: Human embryonic stem cells (hESCs) are pluripotent cells which may differentiate into tissues of all three germ layers. Such research as the feeder-free growth of hESCs is few in China. Fibroblast growth factor (FGF) is a major factor to maintain the undifferentiated state of hESCs.OBJECTIVE: To evaluate the ability of FGF at different concentrations in maintaining the undifferentiated state and pluripotency of hESC lines in the long-term culture.METHODS: Two cell lines of hES-8 and hES-18 were cultured with mouse embryonic fibroblast condition medium for 3 passages and then transferred into mouse embryonic fibroblast condition medium containing different concentrations of FGF: 100, 160, 250 μg/L for 8 passages. The hESCs were removed from the petri dish, cell clusters were digested with collagenase IV and gathered. Cell differentiation and pluripotency were observed. The eighth generation of the hESCs were collected and incubated into severe combined immunodeficiency mice, so as to observe teratoma formation. Morphologies of the cells were evaluated. Alkaline phosphatase staining, surface labeling immunocytochemical analysis and RT-PCR assay method were utilized to determine the OCT-4 expression and tumorigenesis in vivo.RESULTS AND CONCLUSION: Cultured in mouse embryonic fibroblast condition medium containing 160 and 250 μg/L FGF, two cell lines of hESC could maintain undifferentiated state: Clones were round with a high ratio of nucleus to cytoplasm. Large areas in the center of clones were undifferentiated cells, while surrounding the clones were differentiated cells; Strong positive expression for alkaline phosphatase staining was observed; Two cell lines showed high levels of OCT-4 transcription factor protein; The surface markers SSEA-4, TRA-1-60, TRA-1-81 were all positive on both two lines; The hESC clusters could form embryoid body in vivo 10 days later; 3 germ layers of teratomas were also obtained after implanted into severe combined immunodeficiency mice. Mouse embryonic fibroblast condition medium containing 100 μg/L FGF was not sufficient to maintain the long-term proliferation of hESCs, and most of the cells differentiated and died after 4 passages. Alone with concentration 160 μg/LbFGF or more could maintain two hESC lines undifferentiated stably in vitro, has no influence on the differentiation and totipotency of two cell lines.

BACKGROUND: As a main gene engineering vector, adeno-associated virus (AAV) is characterized by its extensive host cells, lasting and stable expression and less immune response to hosts, and is applied widely. But AAV is a kind of defective virus, and need incasing cells to supply E1 protein. As important and special AAV incasing cells, AAV-293 cells can produce E1 in trans. But AAV-293 cells are delicated and cultivated difficultly, and the biological character is easy to be changed. Therefore, it is necessary to establish a culture method of AAV-293 cells to meet the need of gene engineering.OBJECTIVE: To establish a culture method of AAV-293 cells in vitro.DESIGN: An opening study.SETTING: Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: AAV-293 cells line was provided by Stratagene Corporation; high-carbohydrate OMEM (H-DMEM) powder by Gibco Company; there plasmids in AAV Helper-Free by Stratagene Company.METHODS: This experiment was carried out in the neurology laboratory of Tongji Hospital in Wuhan during the period from October 2006 to April 2007. AAV-293 cells were resuscitated and cultivated with H-DMEM growth medium in vitro, and were passaged and stored in liquid nitrogen when the cells monolayer confluence reached 50%. At the same time, their growing state was observed by inverted microscope, and their growth curve was noted. According to whether AAV-293 cells could give out green fluorescence or not (observed by fluorescence inverted microscope) after they were cotransfected with the there AAV system plasmids and infected with AAV supernatant, their biological character of packing AAV was assessed.MAIN OUTCOME MEASURES: ① Morphological observation of AAV-293 cells; ② the growth curve; ③ the package of AAV.RESULTS: ① AAV-293 cells observed by fluorescence inverted microscope were growing adhesively well with irregular polygons, light endochylemas and ambiguous nuclei appearances. ②The growth curve showed that the growing adaptive phase was the first day after AAV-293 cells were passaged, the actively growing phase was from the second day to the fifth day, and the growing platform phase was after the sixth day. ③ AAV-293 cells with green fluorescence observed by fluorescence inverted microscope, and cotransfection of the there AAV system plasmids was successful. AAV-293 cells gave out green fluorescence after infected with AAV supernatant, and AAV package succeeded.CONCLUSION: The culture method established by the authors in the experiment is simple and useful, and the cultured AAV-293 cells remain a good function of AAV package.

The effects of vascular endothelial growth factor (VEGF) on neural differentiation of human embryonic stem cells (hESCs) in vitro and the possible mechanism were observed. The hESCs lines, TJMU1 and TJMU2, were established and stored by our laboratory. hESCs differentiated into neuronal cells through embryonic body formation. In this induction process, hESCs were divided into three groups: group A, routine induction; group B, routine induction+10 ng/mL VEGF; group C, routine induction+10 ng/mL VEGF+10 ng/mL VEGFR2/Fc. OCT4, Nestin and GFAP in each group were detected by RT-PCR, and the cells expressing Nestin and GFAP were counted by immunofluorescence. The percentage of Nestin positive cells in group B was significantly higher than in groups A and C, while the percentage of GFAP positive cells in group B was significantly lower than in groups A and C (P<0.01). There was no significant difference between groups A and C (P>0.05). It was concluded that VEGF, via VEGFR2, stimulated the neural differentiation of hESCs in vitro.

Objective To observe the effect of local mild hypothermia and Naloxone in the treatment of acute intracerebral hemorrhage. Methods Forty-five patients with acute intracerebral hemorrhage were randomly divided into 4 groups:a control group(12 patients),a hypothermia group(11 patients),a Naloxone group(11 patients)and a hypothemrmia plus Naloxone group(11 patients).The patients in the control group were managed with conventional interventions including the administration of 6-aminocaproic acid within 24 hours and dehydrant when intracranial pressure was high.Those in the hypothermia and Naloxone groups were treated with local hypothermia at 33～34 ℃ for 3 days or intravenous transfusion of Naloxone at 4 mg/d in addition to the conventional intervention.Those in the combination group were treated with local hypothermia and intravenous Naloxone in addition to the conventional intervention.Immediately after admission and 2 weeks after treatment,head CT scans were conducted to observe the volume of cerebral hematoma and edema.The patients' neurological function was scored according to the European Stroke Standards(ESS)before and after treatment. Results There was no significant difference among the 4 groups in terms of the volume of hematoma and edema or in their ESS scores before treatment.After treatment,any differences among the 4 groups with regard to hematoma volume were not significant.The volume of edema in the hypothermia group was similar to that in the combination group and significantly lower than that in the Naloxone andcontrol groups.Hematoma volume in the Naloxone group was significantly lower than that in the control group.After treatment,the ESS scores were significantly higher in the combination group than that in hypothermia group,and scores in the hypothermia group were significantly higher than in the Naloxone group.ESS scores in the Naloxone group were significantly higher that in the control group. Conclusion Local mild hypothermia and Naloxone treatment can inhibit cerebral edema and enhance recovery of neurological function in patients with intracerebral hemorrhage.Local mild hypothermia has advantages over Naloxone in inhibiting the development of cerebral edema and in promoting recovery of neurological function.Local mild hypothermia in combination with Naloxone further inhibits edema,and it can enhance neurological function to a greater extent.

Objective To develop transgenic mice harboring the fusion gene of mutant amyloid precursor protein and two types of fluorescent protein for the future study on Alzheimer's disease.Methods The fusion gene CFP-54 bp-YFP-C99 was introduced into mice by mieroinjection.The presence of CFP-54 bp-YFP-C99 was confirmed by PCR in the founders.Results CFP-54 bp-YFP-C99 gene was injected into pronucleus of 2202 zygotes and 1806 injected eggs were implanted into 56 foster mothers, 13 of which were pregnant.There were 13 foster mothers who borne 52 offspring and 32 of them survived.Recipient mouse pregnancy rate was 23.2% (13/56) and the integration rate was 3.9% (2/52).Conclusion CFP-54 bp-YFP-C99 transgenic mice is obtained, but the transgenic efficiency is low.

Objective The results and side effects of nasopharyngeal carcinoma treated by combined external radiotherapy and Californium -252 neutron after loading intracavitary radiotherapy. Methods From November 2005 to March 2007, 30 nasopharyngeal carcinoma patients with staged T1 and T2 by 1992 Fuzhou staging system, were treated by external beam radiotherapy combined with Californium-252 neutron after loading intraeavitary radiotherapy. Results All patients were followed up for 2 years. 2 cases recurred in the nasopharynx. 4 cases developed distant metastases. 2 cases developed trismus. No perforations in hand and soft palate occurred. Conclusion External beam radiotherapy combined with Californian-252 neutron after loading intracavitary radiotherapy for nasopharyngeal carcinoma is indicated for boosting the dose to the nasopharyngeal cavity; reducing the dose for external irradiation. It can improve local control rate of nasopharyngeal carcinoma and reduce occur rate for the trismus.

Objective To study the effects of urinary kallidinogenase (kallikrein) on focal cerebral blood flow (CBF) following cerebral infarction in rats by laser speckle imaging.Methods Permanent middle cerebral artery occlusion (MCAO) was induced in male Sprague-Dawley rats by the intraluminal filament technique.Laser speckle imaging was used to measure CBF in the ischemic cortical area and middle cerebral artery territory.The brain was stained with 2,3,5-triphenyltetrazolium chloride (TTC) to determine the infarct size.Neurological deficit score was measured.Results CBF increased in both hemispheric cortical area and MCA territory on the first and second days following urinary kallikrein administration at high dose but not at low dose.Larger blood vessel diameter and increased blood flow velocity were noticed in the high dose group in some arteries when compared to the low dose group and normal saline control group.At 36 h after cerebral ischemia,the brain infarct size was 10.14% ±3.02% ,25.99% ±3.90% and 27.10% ±3.32% in high, low dose and normal saline control groups,respectively.The infarct size was significantly smaller in the high ( F = 61.14, P<0.01 ) but not low dose group when compared to the normal saline control group.The neurological deficit was milder in the high dose group but not the other two groups at 4 h after cerebral ischemia; however, there were no differences among the groups at 36 h after MCAO.Conclusions Urinary kallidinogenase can reduce cerebral infarction volume and neurological deficit in rats following focal cerebral ischemia.These effects may be attributed to enhanced collateral circulation and improved CBF in the hemispheric cortical area and MCA territory.

Objective To observe senile plaque and iron deposition in cortex and hippocampus of the Alzheimer's disease ( AD ) transgenic mice and investigate their influence on T2 relaxation time.Method All AD transgeic mice were divided into three groups: young group(2,4 months), adult group (6,8,10 months), old group (12,14,16 months), and C57BL/6J mice were as control and were scanned in order by using 4.7 T MR system.Regions of interest (ROI) corresponding to cortex, hippocampus,thalamus, striatum were manually drawn on MR images and T2 MR relaxation times of each ROI were calculated.After MR scan, these mice were decapitated and stained for iron and senile palques.The number of plaque and iron, plaque burden, iron load in cortex and hippocampus were acquired using image pro plus software.Result T2 relaxation times of each group were as following: wild type ( cortex (49.5 ± 2.1 ) ms,hippocampus (51.6 ± 1.1 ) ms ); young ( cortex ( 49.7 ± 0.5 ) ms, hippocampus ( 50.7 ± 0.7 ) ms ); adult (cortex(47.2 ±0.8) ms, hippocampus(47.7 ±0.9) ms) and old (cortex(44.6 ±0.8) ms, hippocampus (45.3 ±0.4)ms).T2 relaxation times in cortex and hippocampus of each group had statistical differences ( cortex F = 18.620, P ＜ 0.01; hippocampus F = 67.925, P ＜ 0.01 ); Compared with young group and wild type mice, T2 relaxation times in corex and hippocampus of adult group mice were decreased significantly.At the same time, T2 relaxation times in old group mice were reduced compared with adult group ( Adult vs young: cortex q =4.284, P ＜0.01, hippocampus q =7.902, P ＜0.01; adult vs wild type: cortex q =4.424, P＜0.05, hippocampus q = 11.450, P ＜0.01; old w adult: cortex q =4.812, P ＜0.01,hippocampus q = 7.034, P ＜ 0.01 ).Histochemical staining for senile plaques found that senile plaques was deposited as early as 4 month.Iron deposition in hippocampus and cortex were detected by perl-DAB as early as 6 months of age, and there was an overall increase in number and load of plaques and iron with age.A positive correlation was observed between plaque burden and iron load ( r = 0.931, P ＜ 0.01 ).At the same time, plaque burden and iron load were negatively correlated with T2 relaxation times ( plaque burden and T2 relaxation times r = - 0.884, P ＜ 0.01; iron load and T2 relaxation times r = - 0.827, P ＜ 0.01 ).Conclusion The changes of T2 relaxation time in AD transgenic mice are attributed to iron and senile plaques.MR T2 relaxation time is a sensitive marker to diagnosis for AD and screen antidementia drugs.

Objective To analyze the present medical teaching textbooks and practice skill guidelines,and explore the profound causes of poor hand hygiene idea among doctors.Methods Three sets of unified textbook series used for domestic medical colleges and universities and two sets of manipulation skill guidelines were studies.Statistical method was conducted to analyze whether concepts and methods of hand hygiene,hand-washing and antiseptic han-drubbing were included in these teaching textbooks;as to eight aseptic manipulation skills,coverage of knowledge, steps of hand-washing and antiseptic handrubbing in manipulation skill guidelines were also analyzed.Results The mentioning rate of hand hygiene,hand-washing and antiseptic handrubbing in 8-year and 5-year program teaching textbooks were both 0 ,in nursing teaching textbooks was 1 00 % ;as to 8 aseptic manipulation in 2 sets of skill practice guidelines,mentioning rate of hand washing was 37 .50 % ,and method and steps of antiseptic handrubbing were both 0 .Conclusion School teaching and skill assessment are the basis,it is difficult to form the right idea by only relying on continuing education without basic education.Hand hygiene should be stressed in the written of teaching textbooks,guidelines should be written following the newest progress,so as to form the correct idea of hand hygiene among doctors.

Objective To study whether methicillin-resistant Staphylococcus aureus (MRSA)will increase the burden of patients with hospital-acquired pneumonia (HAP).Methods Patients with Staphylococcus aureus HAP in a hospital between January 1 ,2013 and November 31 ,2014 were selected,patients with MRSA HAP were as case group,patients with methicillin-sensitive Staphylococcus aureus (MSSA)HAP were as control group,propen-sity score matching (PSM)analysis were conducted to compare the prognosis of MRSA HAP and MSSA HAP (length of hospital stay, duration from infection to discharge, mortality, total therapeutic cost ). Results APACHE II score in case group was higher than control group before PSM was conducted,length of hos-pital stay and duration from infection to discharge were both longer than control group (40[20,94]d vs 28[21 ,53] d;19[10,46]d vs 17[8,29]d,respectively,both P <0.05).APACHE II score in case group and control group af-ter conducting PSM was not significantly different(P >0.05 ),data were balanced and comparable;there were no significant difference in length of hospital stay between two groups (28[21 ,52]d vs 28[21 ,53]d),duration from in-fection to discharge (15[9,25]d vs17[8,29]d),mortality(10.87% vs 15.22%),and total therapeutic cost (121 013.5[80 747.21 ,176 200]yuan vs 119 911 .2[66 994.08,241 184.7]yuan)(all P >0.05).Conclusion APACHE II score is an important factor affecting prognosis;after balancing this factor,there is no difference in the prognosis of patients with MRSA and MSSA HAP,MRSA HAP can not increase the burden of disease.